Allogeneic hematopoietic cell transplantation (allo-HCT) triggers graft vs. leukemia reactions that are uniquely curative for chemotherapy-refractory leukemia. This treatment approach can be made even more effective by enhancing the recovery of the adaptive immune system, thereby reducing the risks of opportunistic infection and relapse. Our long term goals are to make allo-HCT safer, so that more patients can benefit from this procedure. Secondary lymphoid tissues (SLTs) are where antigen-specific immune responses are initiated against pathogens and cancer. Chemotherapy and irradiation, used prior to allo-HCT, damage SLT (including lymph nodes [LN]). This injury delays post-transplant recovery of the adaptive immune system. Methods to repair injured SLTs are urgently needed. Until recently, it was not known how SLTs form during fetal life or how they are remodeled in adult life. Lymphoid tissue inducer (LTi) cells are responsible for fetal LN organogenesis and a related cell type is found in adult human SLT. In mice, adult LTi cells mediate SLT repair. Since LTi cells exist in SLTs, they are extremely difficult to study, especially in humans. As well, ther are considerable differences between murine and human LTi cells, potentially limiting the relevance of murine studies. Nearly all human LTi cell research has used clinical specimens obtained in the setting of pathology. To overcome these issues, we are the only laboratory to have generated LTi cells from human hematopoietic stem cells (HSCs). HSC-derived LTi cells are identical to those isolated from LNs. Using our newly discovered approach, we can generate large numbers of LTi cells for study or potential clinical use. The objectives of this proposal are to further determine the factors involved in LTi differentiation and the mechanisms of LTi-induced SLT repair. In specific aim 1 we will address the hypothesis that stem cell factor (SCF) plays a critical role in HSC commitment to the LTi lineage through the expression of aryl hydrocarbon receptor (AHR). We will also determine the role of soluble and membrane bound SCF on LTi development, survival and expansion. FDA- approved drugs with strong AHR agonist activity will be tested for LTi development or expansion activity. Additional studies will address whether death receptor 3 (DR3) signaling (via TNF ligand 1A (TL1A)) leads to LTi expansion. Our preliminary data shows that LTi cells interact with LN stroma to reorganize into germinal center-like structures and we further hypothesize that LTi cells can restore SLT injury. Lastly, we have found that LTi cells express TNF-superfamily molecules (including OX40L, CD70 LTa1b2 and BAFF) and hypothesize that they costimulate T, B and NK cell activation and proliferation. We will build on these preliminary findings to address whether LTi cells transferred into immunodeficient mice will facilitate human HSC engraftment and/or immune reconstitution. At the conclusion of this project, we will have determined the mechanisms responsible for the commitment of HSCs to the LTi lineage, the factors that lead to LTi cell expansion and whether they facilitate immune recovery in murine models, critical knowledge toward improving outcomes in allo-HCT.